Table 1 Ligands and binding protocols used for receptor autoradiography
From: Multireceptor fingerprints in progressive supranuclear palsy
| Transmitter | Receptor | [3H]-Ligand | Displacer | Incubation buffer | Preincubation | Main incubation | Final rinsea |
|---|---|---|---|---|---|---|---|
| Glutamate | AMPA |
AMPA [10 nM] |
Quisqualate [10 μM] |
50 mM Tris-acetate (pH 7.2) +100 mM KSCNb | 3 × 10 minutes, 4 °C | 45 minutes, 4 °C |
1. 4 × 4 seconds 2. Acetone/glutaraldehyde (100 ml/2.5 ml), 2 × 2 seconds, 4 °C |
| Kainate |
Kainate [9.4 nM] |
SYM 2081 [100 μM] |
50 mM Tris-acetate (pH 7.1) + 10 mM Ca2+-acetate | 3 × 10 minutes, 4 °C | 45 minutes, 4 °C |
1. 4 × 4 seconds, 4 °C 2. Acetone/glutaraldehyde (100 ml/2.5 ml), 2 × 2 seconds, 4 °C | |
| NMDA |
MK-801 [3.3 nM] |
(+)MK-801 [100 μM] |
50 mM Tris-acetate (pH 7.2) + 50 μM glutamate + 30 μM glycineb + 50 μM spermidineb | 15 minutes, 4 °C | 60 minutes, 22 °C | 2 × 5 minutes | |
| mGlu2/3 |
LY341495 [1 nM] |
l-Glutamate [1 mM] |
10 mM phosphate buffer (pH 7.6) + 100 mM KBrb | 2 × 5 minutes, 22 °C | 60 minutes, 4 °C | 2 × 5 minutes | |
| GABA | GABAA |
Muscimol [7.7nM] |
GABA [10 μM] | 50 mM Tris-citrate (pH 7.0) | 3 × 5 minutes, 4 °C | 40 minutes, 4 °C | 3 × 3 seconds |
| GABAB |
CGP 54626 [2 nM] |
CGP 55845 [100 μM] |
50 mM Tris-HCl (pH 7.2) + 2.5 mM CaCl2 | 3 × 5 minutes, 4 °C | 60 minutes, 4 °C | 3 × 2 seconds | |
| BZ |
Flumazenil [1 nM] |
Clonazepam [2 μM] | 170 mM Tris-HCl (pH 7.4) | 15 minutes, 4 °C | 60 minutes, 22 °C | 2 × 1 seconds | |
| pBZ |
PK 11195 [0.1 nM] |
PK 11195 [10 μM] | 50 mM Tris-HCl (pH 7.4) | 15 minutes, 22 °C | 60 minutes, 22 °C | 3 × 5 minutes | |
| Acetylcholine | M1 |
Pirenzepine [1 nM] |
Pirenzepine [2 μM] |
Krebs buffer (pH 7.4) + 4 mM KCl + 120 mM NaCl | 15 minutes, 22 °C | 60 minutes, 22 °C | 3 × 4 minutes |
| M2 |
Oxotremorine-M [1.7 nM] |
Carbachol [10 μM] |
20 mM HEPES-Tris (pH 7.5) + 10 mM MgCl2 + 300 nM pirenzepine | 20 minutes, 22 °C | 60 minutes, 22 °C | 2 × 2 minutes | |
| M3 |
4-DAMP [1 nM] |
Atropine sulfate [10 μM] |
50 mM Tris-HCl (pH 7.4) + 0.1 mM PMSF + 1 mM EDTA | 15 minutes, 22 °C | 45 minutes, 22 °C | 2 × 5 minutes | |
| nACh |
Epibatidine [0.5 nM] |
Nicotine [100 μM] |
1 mM HEPES (pH 7.5) + 120 mM NaCl + 5.4 mM KCl + 0.8 mM MgCl2 + 1.8 mM CaCl2 | 20 minutes, 22 °C | 90 minutes, 22 °C | 5 minutes | |
| Noradrenaline | α1 |
Prazosin [0.2 nM] |
Phentolamine mesylate [10 μM] | 50 mM Na+/K+-phosphate buffer (pH 7.4) | 15 minutes, 22 °C | 60 minutes, 22 °C | 2 × 5 minutes |
| α2 |
UK 14,304 [0.64 nM] |
Phentolamine mesylate [10 μM] |
50 mM Tris-HCl (pH 7.7) + 100 μM MnCl2 | 15 minutes, 22 °C | 90 minutes, 22 °C | 5 minutes | |
| Serotonin | 5-HT1A |
8-OH-DPAT [1 nM] |
5-Hydroxytryptamine [1 μM] |
170 mM Tris-HCl (pH 7.4) + 4 mM CaCl2 b + 0.01% ascorbateb | 30 minutes, 22 °C | 60 minutes, 22 °C | 5 minutes |
| 5-HT2 |
Ketanserin [1.14 nM] |
Mianserin [10 μM] | 170 mM Tris-HCl (pH 7.7) | 30 minutes, 22 °C | 120 minutes, 22 °C | 2 × 10 minutes | |
| Dopamine | D1 |
SCH 23390 [1.67 nM] |
SKF 83566 [1 μM] |
50 mM Tris-HCl (pH 7.4) + 120 mM NaCl + 5 mM KCl + 2 mM CaCl2 + 1 mM MgCl2 | 20 minutes, 22 °C | 90 minutes, 22 °C | 2 × 10 minutes |
| D2 |
Raclopride [0.55 nM] |
Butaclamol [1 μM] |
50 mM Tris-HCl (pH 7.4) + 0.1% ascorbate + 150 mM NaCl | 20 minutes, 22 °C | 45 minutes, 22 °C | 6 × 1 minutes | |
| Adenosine | A1 |
DPCPX [1 nM] |
R-PIA [100 μM] |
170 mM Tris-HCl (pH 7.4) + 2 U/L adenosine deaminase + 100 μM Gpp(NH)pb | 15 minutes, 4 °C | 120 minutes, 22 °C | 2 × 5 minutes |
| A2A |
ZM 241385 [0.42 nM] |
2-Chloroadenosine [20 μM] |
170 mM Tris-HCl (pH 7,4) + 2 U/L adenosine deaminase + 10 mM MgCl2 | 2 × 10 minutes, 22 °C | 120 minutes, 22 °C | 2 × 5 minutes |
